1. Field Of the Invention
The present invention relates to a method of measurement of species which employs enriched isotope spikes in the same speciated form as the species to be measured, equilibration, separation and subsequent determining of concentration by employing isotopic element specie ratios.
2. Description Of the Prior Art
The need for making quantitative determinations of a specie of interest occurs in many environments including environmental, biological, pharmaceutical and industrial samples. For example, certain forms of an element or molecular species may exhibit different toxicities or chemical behaviors from others. Existing techniques, with the exception of electrochemical methods, rely predominately on physical separation in time. They are incapable of determining either cross-over (transformation of one specie form into another), are lost, are altered or are completely recovered. Such techniques cannot be used to determine transformation of one species into another during storage, manipulation and sample preparation or during the measurement process.
An example of the criticality of such measurements would be to consider chromium. While Cr(III) is a trace dement essential for human health, Cr(VI) is poisonous to humans and most other animals and is also a carcinogen. As a result, the difference between these two species, which resides in the oxidation state of the dement, may be of critical importance. While chromatography can be used to separate Cr(III) in time resolution from Cr(VI), as each specie can react with its surroundings and even with separating agencies, the chromatographic separation is only a snapshot in time recording the state of affairs at the end of the manipulation. Each specie may have reacted with many other reagents and transformed during the analysis. There is, therefore, with time resolution, no way of determining how much chromium was actually in each specie when the experiment began or when the sample was actually taken.
Specific species are often required for a particular process. For example, barium is toxic in some compound forms, but is prescribed for medical diagnostic x-ray tests, usually as barium sulfate in liquid slurry form. The conformation and evaluation of a body's processing of barium into another specie can be accomplished with isotopically labeled barium sulfate. These studies have been done, but the use of speciated isotope dilution measurements has not been used for analysis.
Some isotopic tracking has been done for lead due to terrestrially unique and naturally occurring isotopic compositions of this dement. The isotopic ratios can be matched with a particular source to determine the origin of the lead. These measurements are not speciated measurements, but depend on the isotopic ratio differences of the natural material to be detected. This technique has also been used for lead pottery glazes to determine the origin of art objects, however in this case also, naturally occurring isotopic ratios were determined. Lead is a uniquely feasible non-radioactive dement to be evaluated (as to origin) by the isotopic ratio method, as its isotopic ratios change with the amount of uranium mixed with the lead in the original ore deposits. The decay of uranium into different lead isotopes creates unique isotopic ratios for different lead deposits. Uranium also permits this origin-specific ratio identification method.
All elements, with the exception of Pb, U, and Pu, have constant isotopic ratios throughout the earth's crust. Hinners, T. A.; Heithmar, E. M.; Spittier, T. M.; Henshaw, J. M., "Inductively Coupled Plasma Mass Spectrometric Determination of Lead Isotopes" Analytical Chemistry, 59, 2658-2662, 1987. It is this constant elemental isotopic ratio that provides the basis of isotope dilution analysis. When this ratio for an element is artificially altered with an enriched isotope of that element, and bulk analysis of an element is measured from the isotopic ratio, the method is referred to as isotope dilution analysis. The measurement employs a mass spectrometer to determine the isotopic ratio, but only the total elemental concentration is determined. Examples of isotope dilution analysis for total elemental concentration are described for standard reference materials and for general analysis using thermal isotope dilution mass spectrometry. See Moore, Larry J.; Kingston, Howard M.; Murphy, Thomas J.; and Paulsen, Paul J., "The Use of Isotope Dilution Mass Spectrometry for the Certification of Standard Reference Materials", Environment International, 10, 169-173, 1984; Fassett, Jack D. and Paulsen, Paul J., "Isotope Dilution Mass Spectrometry for Accurate Elemental Analysis", Analytical Chemistry, 61, 386-390, 1989. Specific analysis of total chromium and selenium and other metals by this method of isotope dilution mass spectrometry using a gas chromatography mass spectrometer (GC-MS) are described in other references. Reamer, Donald C. and Veillon, Claude, "A Double Isotope Dilution Method for Using Stable Selenium Isotopes in Metabolic Tracer Studies: Analysis by Gas Chromatography/Mass Spectrometry (GC/MS)", Journal of Nutrition, 113, 786-792, 1983. Reamer, Donald C. and Veillon, Claude, "Determination of Selenium in Biological Materials by Stable Isotope Dilution Gas Chromatography-Mass Spectrometry", Analytical Chemistry, 53, 2166-2169, 1981. Veillon, Claude; Wolf, Wayne, and Guthrie, Barbara, "Determination of Chromium in Biological Materials by Stable Isotope Dilution", Analytical Chemistry, 51, 1022-1024, 1979.
Examples of typical prior art are those that use ICP-MS detection with some form of chromatography (sometimes called "flow injection analysis") to separate the species and then uses the instrument as a total elemental detector as described in Thompson, J. J.; Houk, R. S., "Inductively Coupled Plasma Mass Spectrometric Detection for Multielement Flow Injection Analysis and Elemental Speciation by Reversed-Phase Liquid Chromatography" Analytical Chemistry, 58, 2541-2548, 1986. As previously stated herein, in general, when isotope dilution has been used with inductively coupled plasma for environmental analysis, the applications have focused on total analysis and although it is an excellent method for total elemental composition, it has not been effectively applied to species. Examples of ICP-MS isotope dilution analysis for environmental natural water and geological samples are given in Garbarino, H. R.; Taylor, H. E., "Stable Isotope Dilution Analysis of Hydrologic Samples by Inductively Coupled Plasma Mass Spectrometry" Analytical Chemistry, 59, 1568-1575, 1987; McLaren, J. W.; Beauchemin, D.; Berman, S. S., "Application of Isotope Dilution Inductively Coupled Plasma Mass Spectrometry to the Analysis of Marine Sediments" Analytical Chemistry, 59, 610-613, 1987, respectively.
Studies to measure metabolic transformation of elements in humans have been performed where stable isotopes, such as Se-74, have been metabolized in the body and transformed into various species. An enriched isotope is ingested as an inorganic salt and the different forms of the isotope are made by the body. By feeding Se-74 to patients, allowing metabolism, and then removing and storing the blood, previously metabolized Se could be stored for each individual. After the body was free of the labeled Se-74, 11 months later, the blood was reintroduced into the subject and the exact excretion method was studied without the normal body burden of Se confusing the excretion in body fluids. This experiment used stable isotope tracers to studied blood excretion mechanisms and definitively established the dominant pathway to be the urinary path. The studies observed whether body pools could be labeled and how the element is excreted. The metabolic study was specifically for selenium and no specific speciated isotopes or specific species were used to spike in the measurement process. The species were destroyed in the measurement process and total selenium was reported. Following repeated ingestion of an enriched stable isotope of selenium, their blood plasma became labeled with the element in all of the natural, biologically relevant chemical forms. Veillon, Claude; Patterson, Kristine; Button, Lawrence; and Sytkowski, Arthur, "Selenium Utilization in Humans: a Long-Term, Self-Labeling Experiment with Stable Isotopes", American Journal of Clinical Nutrition 52, 155-158, 1990. By spreading the enriched isotope over all species the identification of any specific species becomes impossible by speciated isotope dilution mass spectrometry. "Selenium Utilization in Humans--A Long-Term, Self-Labeling experiment with stable Isotopes", Veillon et at., Am. J. Clin, Nutr.; 52:155-8 (1990) describes this study. Reamer, Donald C. and Veillon, Claude, "A Double Isotope Dilution Method for Using Stable Selenium Isotopes in Metabolic Tracer Studies: Analysis by Gas Chromatography/Mass Spectrometry (GC/MS)", Journal of Nutrition, 113, 786-792, 1983 describes the measurement method of isotope dilution using GC-MS. This method is identical to conventional isotope dilution mass spectrometry as described in Moore, Larry J.; Kingston, Howard M.; Murphy, Thomas J.; and Paulsen, Paul J., "The Use of Isotope Dilution Mass Spectrometry for the Certification of Standard Reference Materials", Environment International, 10, 169-173, 1984; and Fassett, Jack D. and Paulsen, Paul J., "Isotope Dilution Mass Spectrometry for Accurate Elemental Analysis", Analytical Chemistry, 61, 386-390, 1989, except for the type of mass spectrometry equipment used.
Since different species have different reactivates and can react with reagents, container material, oxygen from the air, or other portions of the sample itself, only a final indication of the species separated and detected in time has been employed. The original condition, or the ratio and type of species at the time of sampling or in the sample prior to sampling, cannot be assessed with certainty because transformations or exchanges of the species during storage, chemical manipulation, or the measurement process cannot be measured by conventional species measurement techniques.
Traditional speciation methods rely on separation of the species using physical and/or chemical separation methods. These methods have been summarized in two books. Trace Element Speciation: Analytical Methods and Problems, Ed. Graeme E. Batley, CRC Press, Boca Raton, Fla., 1989 (ISBN 0-8493-4712-2); and Metal Speciation: Theory, Analysis and Application, Eds. James R. Kramer and Herbert E. Allen, Lewis Publishers, Chelsea, Mich., 1991 (ISBN 0-87371-140-8). Neither reference mentions the word "isotope" and no mention of isotopic differentiation, isotopic spike equilibration, or measurement or monitoring using isotopes is mentioned in the speciation literature. Currently, chromatography and other methods of chemical and physical separation are used to perform speciation. Examples of these specific methods are compiled in the Batley and Kramer books, and additional examples are provided.
Aspects of elemental speciation for biological materials are reviewed in Behne, Dietrich, "Speciation of Trace Elements in Biological Materials: Trends and Problems", Analyst, 117, 555-557, 1992. The scope of speciation as related to high performance liquid chromatography can be understood from Cappon, C. J. "HPLC Speciation of Selected Trace Elements" LC-GC, 6, 584-599. 1988.
Early work in chromium speciation prior to spectroscopic equipment is described in Jones, D. R. and Manahan, S. E., "Atomic Absorption Detector for Chromium Organometallic Compounds Separated by High Speed Liquid Chromatography", Analytical Letters, 8, 569-574, 1975. Two current methods for determining chromium species in water, including one commercial version, are described in Lan, Chi-Ren; Tseng, Chia-Liang; Yang, Mo-Hsiung; and Alfassi, Zeer B., "Two-Step Coprecipitation Method for Differentiating Chromium Species in Water Followed by Determination of Chromium by Neutron Activation Analysis", Analyst, 116, 1991; and Determination of Chromium, Application Note 26, Dionex Corporation, May 1986. Energy based separation using microwave extraction is described in Ganzler, K.; Szinai, I.; and Salgo, A., "Effective Sample Preparation Method for Extracting Biologically Active Compounds From Different Matrices by a Microwave Technique", Journal of Chromatography, 520, 257-262, 1990. Most of these methods could be converted to a definitive speciation technique with the addition of spiking with a stable separated isotope specie and use of mass detection following the particular separation method described.
Currently, no definitive method of species measurement is available, although research is needed on many important species of almost every element. In addition to more than 100 elements, literally thousands of species of these elements need to be studied and evaluated. Organic molecules also need investigation. To evaluate the significance and reactions of each, a definitive measurement method is proposed that will work for all elements having more than one isotope. Elements that have radioactive isotopes can also be evaluated. For the examples previously given, chromium has four stable isotopes at constant ratio in nature (Cr-50 at 4.35%, Cr-52 at 83.79%, Cr-53 at 9.50%, and Cr-54 at 2.36%). Within the limits of current isotopic measurement, these ratios are constant throughout the entire earth's crust.
There remains, therefore, a very real and substantial need to provide a method for measuring quantitatively and accurately one or more species within a sample employing speciated isotope dilution measurement and sampling techniques.